Process for the manufacture of butanol or acetone

ABSTRACT

A process for the manufacture of butanol, acetone and other renewable chemicals utilizes one or more of by-products of the manufacture of malt whisky, such as pot ale and spent lees, biomass substrates, such as paper, sludge from paper manufacture and spent grains from distillers and brewers, and diluents, such as water and spent liquid from other fermentations. The process includes treating a substrate to hydrolyze it and fermenting the treated. Also provided is a biofuel including butanol manufactured according to the process.

FIELD OF THE INVENTION

The present invention relates to a process for the manufacture ofbiofuels and renewable chemicals. More particularly, the inventionrelates to a process for the manufacture of butanol. The inventionfurther relates to a process for the manufacture of acetone.

BACKGROUND TO THE INVENTION

In recent years, higher oil prices, depleting fuel supplies andenvironmental concerns have led to a renewed interest in the productionof fuels from biomass (“biofuels”). Biobutanol is produced byfermentation of biomass using bacteria, typically of the genusClostridium. In addition to butanol, these organisms also produceacetone, which is an important solvent, and ethanol. Such a fermentationreaction is often referred to as the “ABE process”(Acetone/Butanol/Ethanol process). Currently used feedstocks orsubstrates include energy crops, such as sugar beets, sugar cane, corngrain and wheat, as well as agricultural by-products, such as straw andcorn stalks. Such substrates provide a balance of nutrients necessary topromote the growth of the organism during fermentation. The use ofbiobutanol as a fuel has several advantages over the use of ethanol.However, as biobutanol production is currently more expensive thanethanol production it has not been commercialized on a large scale.

Many current substrates for the manufacture of butanol and/or acetone byfermentation utilize the energy crops discussed above, which are alreadyin high demand as ingredients for foodstuffs rather than as substratesfor biofuels. A need therefore exists to provide a process for themanufacture of butanol and/or acetone utilizing alternative substrates.

As well as energy crops and agricultural by-products, other organicmaterials, such as organic waste products and/or the organic by-productsof manufacturing processes, may be potential substrate components.However, many prove to be unsuitable. For instance, some organic wasteor by-products may not contain the balance of nutrients suitable topromote the growth of the organism, may be harmful to the organismand/or may not contain sufficient essential nutrients in a formaccessible to the organism. For this reason, many organic waste orby-products are of limited utility as substrates and consequently havelittle economic value.

The present invention provides a process for the manufacture of butanoland/or acetone utilizing a substrate comprising pot ale, a low valueorganic by-product produced as part of the manufacture of whisky,particularly malt whisky.

Malt whisky refers to whisky which has been produced from no grain otherthan malted barley. Production of malt whisky begins with malting ofbarley by steeping the barley in water. Malting releases enzymes thatbreak down starches in the grain and convert them into sugars. When thedesired state of germination is reached, the malted barley is dried. Thedried malted barley is mashed in a mash-tun. In mashing, the enzymesthat were developed during the malting process are allowed to convert orhydrolyse the barley starch into sugar. The resulting liquid whichcontains the sugars is referred to as wort. This is transferred to alarge vessel called a washback where it is cooled and allowed to fermentto form the “wash”. The residue remaining after extraction of thesoluble sugars or wort is known as draff. This comprises spent barleysolids or spent grains.

The wash is distilled in a copper distillation vessel or pot still knownas a wash still to produce an alcohol-containing liquid distillate,known as low wines. The distillation residue or liquor remaining in thepot still after the first distillation of spirit is known as pot ale orburnt ale. The low wines are distilled for a second and sometimes athird time in spirit stills to produce raw spirit, which is matured inoak casks to produce malt whisky. The remaining liquor in the second andsubsequent distillations is called spent lees.

The by-products of the manufacture of malt whisky therefore comprisedraff, pot ale and spent lees. Pot ale has low total solids content andcontains dead yeast cells, yeast residue, soluble protein, solublenutrients, carbohydrates and other material from the fermentation andmashing steps. It can also contain a significant amount of copper fromthe stills themselves. Pot ale is low in nutrients and may have highcopper content, making it a difficulty for disposal within the industry.Significant quantities are disposed of in the sea at a cost to theindustry.

SUMMARY OF THE INVENTION

The inventors of the present application have developed a process forthe manufacture of butanol and/or acetone and optionally other chemicalswhich utilises pot ale, a low or negative economic value by-productobtainable from the manufacture of malt whisky, and a carbohydratesource, such as one obtained from an organic waste product and/or anorganic by-product.

According to a first aspect of the present invention there is provided aprocess for the manufacture of butanol and/or acetone, comprising atleast the steps of:

-   -   treating a substrate comprising a carbohydrate source and pot        ale to solubilise at least a portion of the carbohydrate source        to provide a treated substrate; and    -   fermenting the treated substrate in the presence of a culture of        butanol- and/or acetone-forming micro-organisms at a        concentration of free copper ions of less than 20 μM to provide        a fermented product containing butanol and/or acetone.

In one embodiment, the pot ale is the by-product of the manufacture ofmalt whisky, preferably Scotch malt whisky.

In a further embodiment, the carbohydrate source may include, but is notlimited to, municipal organic waste, industrial organic waste,agricultural crops and crop residues, wood and forestry waste, marinebiomass and bio-energy crops. Thus, the carbohydrate source may beselected from one or more of the group comprising paper, sludge frompaper manufacture, spent grains such as those derived from graindistillers and brewers, fruit and vegetable waste, waste from the bakingindustry, seaweed and seaweed extracts, wood chip and other forestryderivatives, food crops, grain and crop residues, chocolate, algae(macro and micro algae), non-edible crops (and residues) and energycrops, such as switchgrass. According to a preferred embodiment thecarbohydrate source is not malt whisky draff.

In particularly preferred embodiments, the substrate is paper, such aswaste paper. Alternatively the substrate may be food waste and/or wheator wheat-derived substrates such as bread.

In a further embodiment, the fermented product may further comprise oneor more of the compounds selected from the group comprising ethanol,carbon dioxide, hydrogen, acetate and butyrate.

In another embodiment, the substrate may further comprise a diluent. Incertain embodiments, the diluent is selected from one or more of thegroup comprising water, spent lees and spent liquid from otherfermentations. In particular, the diluent may be water.

In a still further embodiment the step of treating the substrate mayfurther comprise:

-   -   diluting the substrate to provide a concentration of free copper        ions of less than 20 μM.

In one embodiment the step of treating the substrate may comprise:

-   -   hydrolysing the carbohydrate source in the presence of water and        hydrogen ions or water and hydroxide ions.

In another embodiment, the step of treating the substrate may comprise:

-   -   hydrolysing the carbohydrate source in the presence of an        aqueous solution of sulphuric acid.

In a further embodiment, the step of treating the substrate does notcomprise heating the substrate at high temperature (i.e. above 120° C.,preferably above 100° C., and more preferably above 80° C.) and/ortreating the substrate with an acid, such as sulphuric acid. Thisparticular embodiment is particularly suitable for the treatment ofpaper or paper-derived products as substrates.

In yet another embodiment, the step of treating of the substratecomprises:

-   -   treating the carbohydrate source with one or more enzymes.

In one embodiment, the step of fermenting the treated substrate may becarried out at a pH of 5.1 or above, more typically in a pH range offrom 5.3 to 5.7, still more typically at a pH of about 5.5. In a furtherembodiment, the pH may be adjusted during the step of fermenting thetreated substrate with a buffer. Alternatively no pH adjustment isrequired, in particular when the substrate is from wood or paper origin.

In another embodiment, the concentration of free copper ions in thefermentation step can be less than 15 μM. In a further embodiment, theconcentration of free copper ions in the fermentation step can bereduced by the addition of a diluent to the treated substrate, such as adiluent described above.

In a still further embodiment, the culture of butanol- and/oracetone-forming micro-organisms may comprise bacteria of the genusclostridium such as: C. acetobutylicum ATCC 824, C. saccharolyticum NCP262, C. beijerinckii NCIMB 8052 and C. saccharoperbutylacetonicum NCIMB12606 (N1-4).

In another embodiment, the fermentation step can carried out without theremoval of solids therefrom.

In further embodiments, the treating and fermenting steps can be carriedout simultaneously or can be carried out sequentially.

In one embodiment, the pot ale may be provided by the steps of:

-   -   adding yeast to a wort comprising water and one or more        carbohydrates selected from the group comprising glucose and        oligosaccharides of glucose;    -   fermenting the wort to provide a wash comprising water and one        or more alcohols; and    -   distilling the wash in a copper distillation vessel to provide a        low wines distillate comprising one or more alcohols and a        distillation residue of pot ale.

In a further embodiment, the wort can be provided by the further stepsof:

-   -   grinding malted barley comprising starch to provide ground        malted barley;    -   mixing the ground malted barley with water to provide a mash        comprising water and ground malted barley;    -   hydrolysing at least a part of the starch in the ground malted        barley of the mash to provide draff comprising spent barley        solids and wort comprising water and one or more carbohydrates        selected from the group comprising glucose and oligosaccharides        of glucose; and    -   separating the draff from the wort.

According to a further aspect of the present invention there is provideda biofuel comprising butanol manufactured according to the process ofany of the aspects of the present invention.

According to a further aspect of the invention there is provided aproduct comprising butanol and/or acetone manufactured according to theprocess of any aspects of the present invention.

According to a yet further aspect of the present invention there isprovided use of pot ale from the production of malt whisky in themanufacture of butanol and/or acetone by fermentation

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) shows sugars resulting from acid and enzyme treatment;

FIG. 1(b) shows residual sugars after fermentation;

FIG. 1(c) shows the ABE products from fermentation;

FIG. 1(d) shows yield of butanol and ABE from draff;

FIG. 2(a) shows ABE production by C. saccharoperbutylacetonicum NCIMB12606 from white office paper;

FIG. 2(b) shows ABE production by C. saccharoperbutylacetonicum NCIMB12606 from newspaper dissolved in either water or 50% pot ale;

FIG. 3 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with white paper solubilised withwater, ammonium acetate or 50% pot ale;

FIG. 4(a) shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia onwhite Paper;

FIG. 4(b) shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia onnewsprint;

FIG. 4(c) shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia onleaflet Paper;

FIG. 5 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia onbread in pot ale;

FIG. 6 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia onwheat in pot ale;

FIG. 7 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with two strains of clostridia onfood waste in pot ale;

FIG. 8 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia inpot ale with wheat that had been subjected to acid treatment; and

FIG. 9 shows the production of acetone, butanol and ethanol fromfermentations (1 litre) carried out with newsprint, white paper andleaflet paper without buffering.

DETAILED DESCRIPTION OF THE INVENTION

The present invention utilises pot ale and a carbohydrate source as asubstrate in the manufacture of butanol and/or acetone by a fermentationprocess. In particular, the present inventors have surprisinglydiscovered that it is possible to carry out fermentation in the presenceof pot ale. It was expected that the high copper content in the pot alefrom the copper pot stills would inhibit butanol- and/or acetone-formingmicro-organisms, such as bacteria of the genus Clostridium. However, thepresent inventors have shown that when the substrate has concentrationof free copper ions to below 20 μM, there is no inhibitory effect.

Pot ale provided direct from the first distillation residue from thecopper pot generally has a concentration of free copper ions at levelswhich are detrimental to butanol- and/or acetone-formingmicro-organisms, such as copper concentrations in excess of 20 μM.Consequently, unmodified pot ale may be an unsuitable medium for formingthe biomass substrate. However, free copper ion concentrations which arenot inhibitory for the butanol- and/or acetone-forming micro-organismscan be achieved by reducing the free copper ion concentration, forinstance by diluting the pot ale, either before it is combined with thecarbohydrate source to provide the substrate, after it has been combinedwith the carbohydrate source to provide the substrate, or after thetreatment of the substrate to provide the treated substrate. However, itwill be apparent that whenever the reduction in free copper ionconcentration is carried out, this should preferably be beforecombination of the treated substrate comprising the pot ale with thebutanol- and/or acetone-forming micro-organisms in order to prevent themicro-organisms being harmed.

The use of pot ale in the manufacture of butanol, acetone and/or otherrenewable chemicals has several associated advantages. Pot ale iscurrently categorised as being of low or negative economic value. Theuse of pot ale in the present invention allows the economic value of potale to be increased. Furthermore, the pot ale acts as a diluent tohydrate the carbohydrate source. Thus, the amount of water or otherdiluent required is reduced when pot ale is used. In addition, pot aleprovides essential nutrients to the microorganisms, particularlyproviding a source of nitrogen which thereby improves the fermentationand overall conversion of substrate to products.

The use of organic waste or by-products as the carbohydrate source inthe substrate for the manufacture of butanol and/or acetone andoptionally other chemicals is also advantageous as it provides asolution to the disposal of these substances. In certain aspects, thepresent invention utilises paper, and in particular waste paper, as acarbohydrate source. The present invention therefore further provides asolution to the disposal of waste paper, for example, old newspapers orused photocopier paper. It should be noted that the use of pot ale hasbeen found necessary to create a suitable fermentation medium for theproduction of butanol and/or acetone from paper based substrates.

The substrate must be treated to solubilise at least a part of thecarbohydrate source. The solubilisation may take the form of hydrolysis,thus breaking down the carbohydrate source into a form suitable forfermentation. Accordingly, in certain embodiments the substrate issubjected to one or more treatment steps to solubilise and particularlyhydrolyse the carbohydrate source, for example, mashing, heating,addition of acid or alkali, addition of enzymes or a combinationthereof. In certain embodiments, the treating of the substrate tosolubilise the carbohydrate source comprises the step of hydrolysing thecarbohydrate source in the presence of water and hydrogen ions or waterand hydroxide ions. In certain embodiments, the treating of thecarbohydrate source to solubilise it is carried out in the presence ofany suitable acid which is capable of hydrolysing the carbohydratesource. Examples of suitable acids include sulphuric acid and nitricacid. Sulphuric acid is a preferred example of an acid for use in theprocess described herein. When the substrate is paper based, or paperderived, no temperature or acid treatment is required.

In certain embodiments, the treating of the substrate to hydrolyse thecarbohydrate source comprises addition of one or more enzymes, such ascellulase and hemicellulase. In certain embodiments, a combination oftreatments may be utilised, for example, addition of both acid andenzymes, to provide a treated substrate in a form suitable forfermentation. The combination of treatments may be appliedsimultaneously or sequentially.

In another embodiment wherein the substrate comprises at least one ofpaper, paper waste, wheat and wheat based products such as bread, thetreatment may comprise the addition of enzymes. This enzyme treatmentmay occur at a temperature lower than that which is optimal for enzymeactivity (i.e. about 50° C.). More specifically the temperature may bethe same as the fermentation temperature (e.g. 33° C.), allowing bothprocesses to proceed simultaneously. Suitable temperature ranges can be25 to 40° C., preferably 30 to 35° C., and more preferably 32 to 34° C.Only enzyme treatment may be required because steps earlier in themanufacture of products such as bread and/or paper waste can concentratethe carbohydrate content and remove inhibitory materials (e.g. ligninfrom paper based products).

Fermentation of the treated substrate is carried out at pH in the rangeof 5.1 or above, more preferably in the range of from 5.2 to 6.2, stillmore preferably in the range of from 5.3 to 5.7 and yet more preferablyat about 5.5. The use of the pH range of from 5.3 to 5.7 has been shownto provide high yields of butanol and/or acetone. Furthermore, this pHrange allows fermentation to be carried out without the need to removesolids therefrom, thus reducing costs and avoiding any technicalproblems caused by the requirement to remove solids. This pH rangeprevents any potential toxicity from the treated substrate whilemaximising butanol and/or acetone production.

Fermentation is carried out in the presence of a culture of butanol-and/or acetone-forming micro-organisms. The butanol- and/oracetone-forming micro-organisms may be selected from any solventproducing micro-organisms which are capable of fermenting the substrateto form butanol and/or acetone. Suitable micro-organisms includemicro-organisms engineered to produce solvents. Examples of suitablemicro-organisms include those currently used in ABE(Acetone/Butanol/Ethanol) manufacture, and, in particular, bacteria ofthe genus clostridium such as C. acetobutylicum, C. beijerinckii, C.saccharoperbutylacetonicum and C. saccharobutylicum. In someembodiments, the butanol- and/or acetone forming micro-organismscomprise C. acetobutylicum. In other embodiments the micro-organismscomprises C. Saccharoperbutylacetonicum. This particular strain isparticularly effective on paper-based substrates.

Fermentation is carried out at a concentration of free copper ions ofless than 20 μM. This ensures that the presence of the copper ions haveno/minimal negative effect. In certain embodiments, water or anotheraqueous solution may be added to lower the concentration of free copperions to below 20 μM free copper ions. In certain embodiments, theconcentration of free copper ions is less than 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6 or 5 μM free copper ions during at least thefermentation step. In certain embodiments, the concentration of freecopper ions is less than 15 μM. In certain embodiments, theconcentration of free copper ions is less than 10 μM.

In certain embodiments, the treating and fermenting steps are carriedout simultaneously. This reduces the amount of time required, the numberof steps involved and the associated cost of manufacture.

In alternative embodiments, the treating and fermenting steps arecarried out sequentially. For example, the substrate may be pre-treatedin two steps, first with acid and subsequently with enzyme, prior tofermentation.

In certain embodiments, the fermented product further comprises one ormore of the compounds selected from the group comprising ethanol, carbondioxide, hydrogen, acetate and butyrate. Butanol and/or acetone may beseparated out of the fermented product using conventional separationtechniques. Alternatively, the fermented product may be used as a fuelor otherwise without further purification.

The term “biobutanol” as used herein refers to butanol made frombiomass.

The term “pot ale” as used herein refers to the liquor remaining in thewash (copper pot) still after the first distillation in the manufactureof malt whisky. It is the residue of the wash after extraction bydistillation of the low wines.

The term “spent lees” as used herein refers to the liquor remaining inthe distillation vessel after second and subsequent distillations in themanufacture of malt whisky. It is the residue of the low wines afterextraction by distillation of raw spirit.

The term “concentration of free copper ions” refers to the concentrationof copper ions which is not bound to solids, that is, the concentrationof copper ions in the supernatant. The total concentration of copper inthe pot ale will be higher than the concentration of free copper ions assome copper remains bound to solids, such as dead yeast cells.

The term “Scotch whisky” as used herein refers to whisky made inScotland. In alternative embodiments, the malt whisky is a malt whiskymanufactured in other countries, such as Ireland or India, where theprocess for manufacture of malt whisky in that country is similar oridentical to the process used in Scotland for the manufacture of Scotchmalt whisky.

The present invention will now be described with reference to thefollowing examples which are provided for the purpose of illustrationand are not intended to be construed as being limiting on the presentinvention.

FIG. 1 shows the influence of initial pH on fermentation of acid andenzyme pre-treated draff in pot ale by C. acetobutylicum ATCC 824. Draffwas pre-treated with 0.08 M H₂SO₄ and the pH adjusted to between pH5.0-6.0 prior to enzyme addition. After enzyme hydrolysis, the pH wasadjusted to 4.5, 4.8, 5.0, 5.5, 6.0 or 6.5 for fermentation. FIG. 1 (a)shows sugars resulting from acid and enzyme treatment, FIG. 1 (b) showsresidual sugars after fermentation, FIG. 1 (c) shows the ABE productsfrom fermentation and FIG. 1 (d) shows yield of butanol and ABE fromdraff;

FIG. 2 shows ABE production by C. saccharoperbutylacetonicum NCIMB 12606from (a) white office paper and (b) newspaper dissolved in either wateror 50% pot ale.

FIG. 3 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with white paper solubilised withwater, ammonium acetate or 50% pot ale.

FIGS. 4a to 4c show the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia on a)white Paper, b) newsprint and c) leaflet Paper. Substrates were dilutedin 50% pot ale, buffered to pH 5.5 and treated with industrial enzymes.

FIG. 5 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia onbread in pot ale.

FIG. 6 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia onwheat in pot ale.

FIG. 7 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with two strains of clostridia onfood waste in pot ale.

FIG. 8 shows the production of acetone, butanol and ethanol fromfermentations (150 ml) carried out with four strains of clostridia inpot ale with wheat that had been subjected to acid treatment.

FIG. 9 shows the production of acetone, butanol and ethanol fromfermentations (1 litre) carried out with newsprint, white paper andleaflet paper without buffering.

EXAMPLES

Reference Example 1 and Example 2 and 3 show the effect of pH and freecopper ion concentration of the substrate upon the fermentation process.In both of Reference Example 1 and Example 3, glucose was used as thecarbohydrate source. Reference Example 1 is designated as a referencetest because no pot ale was present in the substrate.

Reference Example 1 shows that ABE can be produced from the fermentationof glucose in a pH range of above 4.5 to 6.5. At a pH of 4.5 no glucosewas consumed or ABE produced. At a pH of 6.5, only acids were produced,with no ABE. Example 2 shows a preferred initial range pH of about 5.5for the fermentation of a substrate comprising pot ale and a substrate(draff).

Example 3 shows that undiluted pot ale has a concentration of freecopper ions which is detrimental to fermentation, resulting in areduction in ABE yield compared to a substrate without added free copperions. Furthermore as the concentration of free copper ions was reducedfrom 20 μM to 5 μM, the yield of ABE increased to that obtained from asubstrate with no added free copper ions.

Example 4 shows that the addition of pot ale to a substrate results inan increase in ABE yield, compared to the addition of a diluent such aswater.

Example 5 shows that the addition of pot ale to a substrate has the sameeffect as the addition of ammonium sulphate, indicating the pot ale actsas a nitrogen source.

Example 6 shows that ABE can be produced using pot ale in combinationwith various substrates and microorganisms. The substrates exemplifiedare various types of paper material, bread, wheat and food waste.

Example 7 shows that certain substrates do not require acid treatment.Indeed acid treatment of wheat produces lower yields of ABE thanuntreated wheat.

Example 8 shows that pre-fermentation buffering is not always requiredon paper based substrates.

General Methods

The following organisms were used: C. acetobutylicum ATCC 824, C.saccharolyticum NCP 262, C. beijerinckii NCIMB 8052 and C.saccharoperbutylacetonicum NCIMB 12606 (N1-4). Clostridia weremaintained as spore suspensions at 4° C. Spores were heat shocked at 80°C. for 10 minutes and inoculated into reinforced clostridia media (RCM,Oxoid Ltd, Cambridge, UK).

In examples 1 to 3 the cultures were incubated for 24 hours and thensubcultured into tryptone-yeast extract-ammonium acetate media (TYA)media containing glucose before being used as a starting culture (at 5%v/v) for all experiments. TYA consisted of (g/l) tryptone, 6; yeastextract, 2; ammonium acetate, 3; KH₂PO₄, 0.5; MgSO₄.7H₂O, 0.3;FeSO₄.7H₂O, 0.01 supplemented with 5% glucose. All clostridia cultureswere incubated in an anaerobic workstation under an N₂—H₂—CO₂ (80:10:10)atmosphere at 33° C. In examples 4 to 8 starter cultures were incubatedfor 24 h and then subcultured into 50% pot ale buffered to pH 5.5. Allclostridia cultures were incubated in an anaerobic workstation under anN₂—H₂—CO₂ (80:10:10) atmosphere at 33° C. Small scale fermentation (150ml) was carried out in glass bottles in the anaerobic workstation.

For 1 L scale, fermentations were conducted in fermenters (Biostat APlus, Sartorius Stedim Ltd, Surrey, UK). Oxygen-free conditions wereachieved by sparging the media in the fermenters with oxygen-free N₂ for1 hour prior to inoculation with clostridia. For all 1 L fermentations,agitation was set at 200 rpm and temperature at 33° C.

Pot ale was obtained from distilleries and was diluted in a 1:1 ratio byvolume with water. This was sufficient to reduce the concentration offree copper ions to less than 20 μM.

Wet draff, as received from the distilleries, had a moisture contentbetween 75-80%. Where stated, draff was dried at 80° C. to a moisturecontent of approximately 4% and milled prior to further processing.

Ethanol, Acetone and Butanol were measured using a Chrompack 9001 gaschromatograph equipped with a flame ionisation detector and a CP SIL 5CBcolumn of length 10 m and diameter 0.32 mm (all Chrompack, Middelburg,Netherlands). All samples were filtered through 0.2 μm cellulose acetatesyringe filters before analysis and concentrations were determined byreference to ethanol, acetone and butanol standards.

For acid (acetic and butyric) and monosaccharide (glucose, xylose andarabinose) analysis, samples were filtered through 0.2 μm syringefilters and acidified with H₂SO₄. Samples were analysed by HPLC using aVarian 920 LC fitted with integrated UV-VIS dual wavelength andrefractive index detectors (Varian Ltd., Oxford, UK). Components wereseparated at room temperature on a Rezex ROA Organic acid H⁺ 8% 300×7.8mm column (Phenomenex, Cheshire, UK) with 0.005 N H₂SO₄ as the mobilephase at a flow rate of 0.5 ml/min. Acids were detected at 210 nm whilesugars were detected with the RI detector and concentrations weredetermined by reference to the corresponding standards.

Reference Example 1—Effect of pH Control on ABE Production by Clostridia

The effect of pH on fermentation of glucose in TYA media by C.acetobutylicum ATCC 824 was investigated. Fermentations were conductedat 1 L scale and the pH was controlled at a range of set points betweenpH 4.5-6.5 with automated addition of either alkali or acid. At pH 4.5,no glucose utilisation, acid or ABE production was detected. For allother fermentations, glucose was completely consumed within 48 hours andacids (butyric and acetic) and solvent (acetone, butanol and ethanol)were produced (Table 2). ABE production was highest at pH 4.8 and 5.0,corresponding to yields of 0.34 and 0.30 g ABE/g sugar, respectively.Acid production increased between pH 5.5 to 6.5, with a correspondingdecrease in conversion of sugar to ABE. At pH 6.5, acids only wereproduced with final concentrations of 7.8 and 12.8 g/l acetic andbutyric acid, respectively.

TABLE 1 Conversion of 5% glucose to acid and ABE by C. acetobutylicumATCC 824 in TYA media controlled at either pH 4.8, 5.0, 5.5, 6.0 or 6.5.Acid (butyric and acetic) and ABE concentrations were determined after68 hours with ABE yield expressed as g of ABE produced per g of sugarconsumed. pH Acid (g/l) ABE (g/l) Yield (g ABE/g sugar) 4.8 0.7 15.20.34 5.0 0.9 14.3 0.30 5.5 7.9 12.3 0.25 6.0 13.6 6.7 0.13 6.5 20.5 0.60.01

Example 2—Influence of Initial pH on ABE Production by Clostridia

The effect of initial pH on fermentation of a pre-treated substratecomprising draff and pot ale was investigated. Dried, milled draff waspre-treated by adding 10.5% (w/v) to 250 ml duran bottles with 0.08 MH₂SO₄ in 50% pot ale and sterilised at 121° C. for 15 min. Aftercooling, the pH was adjusted to between pH 5.0-6.0 by addition of 10 MNaOH and incubated with cellulase and hemicellulase enzymes at 33° C.for 24 hours. For fermentation, the initial pH of the solutions wasadjusted to either 4.5, 4.8, 5.0, 5.5, 6.0 or 6.5 prior to inoculationwith C. acetobutylicum ATCC 824. The initial sugar concentration wasmonitored before fermentation and the residual sugar, ABE concentrationand ABE yield were calculated after fermentation (FIG. 1). The initialconcentration of sugars was similar for all samples, with approximately9.6, 11.2, and 9.9 g/l glucose, xylose and arabinose. No growth or gasproduction was apparent at pH 5.0 or lower and no sugars were utilised.ABE production was greatest at pH 5.5 (14.2 g/l) with a yield of 13.2g/100 g draff. This was reduced at pH 6.0, with 9.3 g ABE/100 g draff.At pH 6.5, approximately half the sugar was utilised but there was poorconversion to ABE with a final concentration of 2.3 g/l.

Example 3—Pot Ale as a Growth Medium for ABE Production by Clostridia

Pot ale was collected from a Scottish malt distillery and analysed forcopper content. The pot ale had 71.8 μM total Cu of which 21.1 μM wasdetermined to be available as “free” Cu in the supernatant with the restbound to the solids. To assess whether this Cu concentration was toxicto C. acetobutylicum ATCC 824, fermentation of 5% glucose in 100 ml TYAmedia supplemented with different concentrations of Cu was compared(Table 3). Cu had no effect on ABE production at 5 and 10 μM with ABEconcentrations of approximately 12 g/l being similar to that of thecontrol without Cu. At the higher Cu concentration, ABE concentrationwas reduced to 8.6 g/l, indicating that at this concentration Cu wasinhibitory to clostridia. As the pot ale had a “free” Cu content of 21.1μM, it was decided to test clostridia fermentation in half strength potale in order to reduce the Cu concentration below inhibitory levels.Half-strength pot ale supplemented with glucose provided enoughnutrients for growth of 824 with ABE production similar to the TYAcontrol (Table 3).

TABLE 2 Conversion of 5% glucose to ABE by C. acetobutylicum ATCC 824 ineither TYA, TYA containing 5, 10 or 20 μM Cu or 50% pot ale. Media ABE(g/l) TYA 12.4 ± 0.3 TYA, 5 μM Cu 12.3 ± 0.3 TYA, 10 μM Cu 11.6 ± 0.1TYA 20 μM Cu  8.6 ± 2.0 50% pot ale 12.0 ± 1.7

Example 4—Process for Conversion of Waste Paper to Butanol and Acetone

White office paper and newspaper were shredded to 5 mm wide strips and6.7% (w/v) was mixed with either water or 50% pot ale in 250 ml duranbottles and the pH adjusted to pH 5.5. After sterilisation, the bottleswere cooled and cellulase and C. saccharoperbutylacetonicum NCIMB 12606added. After fermentation, the ABE concentrations were determined (FIG.2). There was poor conversion of paper to ABE in water compared to potale, demonstrating that pot ale was required to provide additionalnutrients. In pot ale, the ABE yields after fermentation with C.saccharoperbutylacetonicum were 24.8 g ABE per 100 g office paper and16.8 g ABE per 100 g newspaper.

Example 5—Effect of Pot Ale as a Nitrogen Source on ABE Production byClostridia

Certain substances which have had their carbohydrate content increasedby processing may be deficient in nitrogen. A substrate comprising paperas a carbohydrate source and 50% pot ale was compared to a substratecomprising paper and water and a substrate comprising paper and aqueousammonium acetate. The modus operandi used was the same than the one usefor the previous example.

As shown in FIG. 3, fermentation of the substrate comprising paper andwater produced little ABE. Supplementing a substrate comprising paperand water with ammonium acetate produced good results, indicating that apaper carbohydrate source was deficient in nitrogen. The substratecomprising paper and pot ale produced even better results as thesubstrate supplemented with ammonium acetate. FIG. 3 shows thatfermentations using either ammonium sulphate or pot ale produced moreABE than those using water. The strain used was N1-4.

Example 6—Production of ABE from a Range of Substrates Comprising PotAle

A range of substrates comprising a carbohydrate source were selectedfrom the group comprising white paper, newsprint, leaflet paper, bread(white from Kingsmill), wheat and food waste. The first fivecarbohydrate sources represent readily available waste materials, whilewheat represents an alternative agricultural crop. The carbohydratesources were prepared as follows: paper samples were shredded using acommercial paper shredder, the bread, wheat and food waste samples wereshredded in a domestic food processor. The carbohydrate sources weresolubilised in 50% pot ale, buffered at pH 5.5 with sodium hydroxide,treated with industrial enzymes (C-Tech and H-Tech, commerciallyavailable cellulase and hemicellulase from Novozymes) and fermented withthe four strains of clostridia bacteria previously mentioned. All thefermentations produced ABE (shown in FIGS. 4-7). The N1-4 strain wasparticularly efficient with respect to the paper based substrates as theABE concentration produced was consistently above 7 g/l and can even beabove 12 g/l.

Example 7—Pre-Treatment of Different Carbohydrate Sources

Malt whisky draff requires treatment with acid at high temperature torender its structure accessible to the enzymes used to release sugarsfor fermentation. In contrast, carbohydrate sources such as bread andpaper wastes do not require treatment as processes carried out earlierin the manufacturing process have removed inhibitory materials such aslignin and concentrated the carbohydrate content. Thus, suchcarbohydrate sources require only enzyme treatment at low temperaturebefore fermentation.

FIG. 8 shows the concentration of ABE obtained after wheat is subjectedto an acid treatment. As it can be seen when compared with the resultsshown in FIG. 6, when used on wheat, the treatment does not lead to anyimprovements and can even show severe detrimental effects with respectto particular strains of clostridia (i.e. P262 and N1-4).

Example 10—Pre-Fermentation Buffering

Those substrates that have not undergone acid treatment have higher pHvalues than those that have undergone acid treatment. Thus, buffering tothe fermentation pH in those substrates which have not undergone acidtreatment may be less intensive and in some cases is not required. Asshown in FIG. 9, when paper is used as a carbohydrate source, bufferingwas not required because the optimal strain, N1-4, can ferment at ahigher pH such that satisfactory performance was achieved withoutbuffering before inoculation.

Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes of carrying out theinvention which are obvious to those skilled in the art are intended tobe covered by the present invention.

1-19. (canceled)
 20. A process for the manufacture of butanol and/oracetone, the process comprising: treating a substrate comprising acarbohydrate source and pot ale to solubilise at least a portion of thecarbohydrate source to provide a treated substrate; and fermenting thetreated substrate in the presence of a culture of butanol- and/oracetone-forming micro-organisms at a concentration of free copper ionsof less than 20μ to provide a fermented product containing butanoland/or acetone.
 21. The process as claimed in claim 20, wherein the stepof treating the substrate comprises: hydrolysing the carbohydrate sourcein the presence of water and hydrogen ions or water and hydroxide ions.22. The process as claimed in claim 20, wherein the step of treating thesubstrate comprises: hydrolysing the carbohydrate source in the presenceof an aqueous solution of sulphuric acid.
 23. The process as claimed inclaim 20, wherein the step of treating of the substrate comprisestreating the carbohydrate source with one or more enzymes.
 24. Theprocess as claimed in claim 20, wherein the step of treating thesubstrate comprises diluting the substrate to provide a concentration offree copper ions of less than 20μ.
 25. The process as claimed in claim20, wherein the step of fermenting the treated substrate is carried outat a pH of 5.1 or above.
 26. The process as claimed in claim 20, whereinthe concentration of free copper ions in the fermentation step is lessthan 15μ.
 27. The process as claimed in claim 20, wherein the culture ofbutanol- and/or acetone-forming micro-organisms comprises bacteria ofthe genus Clostridium.
 28. The process as claimed in claim 20, whereinthe fermentation step is carried out without the removal of solidstherefrom.
 29. The process as claimed in claim 20, wherein the treatingand fermenting steps are carried out simultaneously.
 30. The process asclaimed in claim 20, wherein the treating and fermenting steps arecarried out sequentially.
 31. The process as claimed in claim 20,wherein the pot ale is provided by the steps of: adding yeast to a wortcomprising water and one or more carbohydrates selected from the groupcomprising glucose and oligosaccharides of glucose; fermenting the wortto provide a wash comprising water and one or more alcohols; anddistilling the wash in a copper distillation vessel to provide a lowwines distillate comprising one or more alcohols and a distillationresidue of pot ale.
 32. The process as claimed in claim 31, wherein thewort is provided by the further steps of: grinding malted barleycomprising starch to provide ground malted barley; mixing the groundmalted barley with water to provide a mash comprising water and groundmalted barley; hydrolysing at least a part of the starch in the groundmalted barley of the mash to provide draff comprising spent barleysolids and wort comprising water and one or more carbohydrates selectedfrom the group comprising glucose and oligosaccharides of glucose; andseparating the draff from the wort.
 33. The process as claimed in claim20, wherein the pot ale is the by-product of the manufacture of maltwhisky.
 34. The process as claimed in claim 20, wherein the carbohydratesource is selected from one or more of the group comprising paper,sludge from paper manufacture, fruit and vegetable waste, waste from thebaking industry, seaweed and seaweed extracts, forestry derivatives,food crops, grain and crop residues, spent grains, chocolate, algae,non-edible crops and their residues and energy crops.
 35. The process asclaimed in claim 20, wherein the fermented product further comprises oneor more of the compounds selected from the group comprising ethanol,carbon dioxide, hydrogen, acetate and butyrate.
 36. A biofuel comprisingbutanol manufactured according to the process of claim 20.